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Recent studies have shown that stem cell memory T (TSCM) cell-like properties are important for successful adoptive immunotherapy by the chimeric antigen receptor–engineered-T (CAR-T) cells. We previously reported that both human and murine-activated T cells are converted into stem cell memory-like T (iTSCM) cells by coculture with stromal OP9 cells expressing the NOTCH ligand. However, the mechanism of NOTCH-mediated iTSCM reprogramming remains to be elucidated. Here, we report that the NOTCH/OP9 system efficiently converted conventional human CAR-T cells into TSCM-like CAR-T, “CAR-iTSCM” cells, and that mitochondrial metabolic reprogramming played a key role in this conversion. NOTCH signaling promoted mitochondrial biogenesis and fatty acid synthesis during iTSCM formation, which are essential for the properties of iTSCM cells. Forkhead box M1 (FOXM1) was identified as a downstream target of NOTCH, which was responsible for these metabolic changes and the subsequent iTSCM differentiation. Like NOTCH-induced CAR-iTSCM cells, FOXM1-induced CAR-iTSCM cells possessed superior antitumor potential compared with conventional CAR-T cells. We propose that NOTCH- or FOXM1-driven CAR-iTSCM formation is an effective strategy for improving cancer immunotherapy.Significance:Manipulation of signaling and metabolic pathways important for directing production of stem cell memory–like T cells may enable development of improved CAR-T cells.

Supplementary Figure S1. Metabolic analysis before or after the OP9-hDLL1 co-culture. Supplementary Figure S2. Metformin disrupts iTSCM induction Supplementary Figure S3. Anti-CD19 CAR-iTSCM cells possess superior antitumor ability in B-ALL models. Supplementary Figure S4. The effects of Notch intracellular domain(NICD) overexpression on T cell phenotypes. Supplementary Figure S5. NICDÎ"ANK-overexpression does not induce iTSCM cells Supplementary Figure S6. The regulation of FOXM1 expression by NICD or FOXM1 knockout. Supplementary Figure S7. FOXM1â^†N-overexpression induces iTSCM formation Supplementary Figure S8. NICD- and FOXM1-overexpressed TSCM-like cells are close to iTSCM cells induced by OP9-hDLL1 cells.

T cells with a stem cell memory (TSCM) phenotype provide long-term and potent antitumor effects for T-cell transfer therapies. Although various methods for the induction of TSCM-like cells in vitro have been reported, few methods generate TSCM-like cells from effector/exhausted T cells. We have reported that coculture with the Notch ligand–expressing OP9 stromal cells induces TSCM-like (iTSCM) cells. Here, we established a feeder-free culture system to improve iTSCM cell generation from expanded chimeric antigen receptor (CAR)-expressing T cells; culturing CAR T cells in the presence of IL7, CXCL12, IGF-I, and the Notch ligand, hDLL1. Feeder-free CAR-iTSCM cells showed the expression of cell surface markers and genes similar to that of OP9-hDLL1 feeder cell–induced CAR-iTSCM cells, including the elevated expression of SCM-associated genes, TCF7, LEF1, and BCL6, and reduced expression of exhaustion-associated genes like LAG3, TOX, and NR4A1. Feeder-free CAR-iTSCM cells showed higher proliferative capacity depending on oxidative phosphorylation and exhibited higher IL2 production and stronger antitumor activity in vivo than feeder cell–induced CAR-iTSCM cells. Our feeder-free culture system represents a way to rejuvenate effector/exhausted CAR T cells to SCM-like CAR T cells. Significance: Resting CAR T cells with our defined factors reprograms exhausted state to SCM-like state and enables development of improved CAR T-cell therapy.

Recent studies have shown that stem cell memory T (TSCM) cell-like properties are important for successful adoptive immunotherapy by the chimeric antigen receptor–engineered-T (CAR-T) cells. We previously reported that both human and murine-activated T cells are converted into stem cell memory-like T (iTSCM) cells by coculture with stromal OP9 cells expressing the NOTCH ligand. However, the mechanism of NOTCH-mediated iTSCM reprogramming remains to be elucidated. Here, we report that the NOTCH/OP9 system efficiently converted conventional human CAR-T cells into TSCM-like CAR-T, “CAR-iTSCM” cells, and that mitochondrial metabolic reprogramming played a key role in this conversion. NOTCH signaling promoted mitochondrial biogenesis and fatty acid synthesis during iTSCM formation, which are essential for the properties of iTSCM cells. Forkhead box M1 (FOXM1) was identified as a downstream target of NOTCH, which was responsible for these metabolic changes and the subsequent iTSCM differentiation. Like NOTCH-induced CAR-iTSCM cells, FOXM1-induced CAR-iTSCM cells possessed superior antitumor potential compared with conventional CAR-T cells. We propose that NOTCH- or FOXM1-driven CAR-iTSCM formation is an effective strategy for improving cancer immunotherapy. Significance: Manipulation of signaling and metabolic pathways important for directing production of stem cell memory–like T cells may enable development of improved CAR-T cells.

The pharmacokinetic (PK) and pharmacodynamic (PD) modeling was conducted for the reduction of peripheral lymphocytes after oral administration of CS-0777 to healthy rats, monkeys and experimental autoimmune encephalomyelitis (EAE) induced rats. M1, the phosphorylated active metabolite of CS-0777, is a selective sphingosine 1-phosphate receptor-1 modulator. A linear one-and two-compartment model with reversible metabolism process characterized the time courses of CS-0777 and M1 concentrations in rats and monkeys, respectively. The relationship between lymphocyte counts and M1 concentrations in blood was well described via an indirect response model in all animals examined. An Imax of 0.815 and an IC50 of 6.58 ng/ml in healthy rats, an Imax of 0.807 and an IC50 of 5.09 ng/ml in the EAE rats, an Imax of 0.789 and an IC50 of 0.484 ng/ml in monkeys were estimated by indirect PD model. Since the IC50 values calculated in terms of an unbound plasma concentration-basis in rats and monkeys were within the similar range by correcting the IC50 in blood described above with blood to plasma concentration ratio and plasma free fraction of M1 in each animal, it is considered that the intrinsic activity of M1 against lymphocyte reduction exhibits no species difference. The sensitivity of the lymphocytes against M1 was not affected by the status of EAE. The comparison of the simulated lymphocyte reduction in EAE rats by multiple dosing of CS-0777 and actual EAE clinical scores implies that to show significant suppressive effect on EAE does not require absolute elimination of all lymphocytes from the blood.

The recessive N-ethyl-N-nitrosourea-induced phenotype toku is characterized by delayed hair growth, progressive hair loss, and excessive accumulation of dermal cholesterol, triglycerides, and ceramides. The toku phenotype was attributed to a null allele of Gk5, encoding glycerol kinase 5 (GK5), a skin-specific kinase expressed predominantly in sebaceous glands. GK5 formed a complex with the sterol regulatory element-binding proteins (SREBPs) through their C-terminal regulatory domains, inhibiting SREBP processing and activation. In Gk5toku/toku mice, transcriptionally active SREBPs accumulated in the skin, but not in the liver; they were localized to the nucleus and led to elevated lipid synthesis and subsequent hair growth defects. Similar defective hair growth was observed in kinase-inactive GK5 mutant mice. Hair growth defects of homozygous toku mice were partially rescued by treatment with the HMG-CoA reductase inhibitor simvastatin. GK5 exists as part of a skin-specific regulatory mechanism for cholesterol biosynthesis, independent of cholesterol regulation elsewhere in the body.

Significance Whereas many heritable obesity phenotypes are known, lean phenotypes are comparatively uncommon. Yet they can reveal critical checkpoints regulating energy balance. During a large-scale random germ-line mutagenesis project, we identified mice with a lean phenotype, myopathy, excessive energy expenditure despite diminished cage activity, and impaired glucose tolerance. This phenotype, termed “ supermodel, ” was strictly recessive and was ascribed to a missense mutation in Sterile alpha motif domain containing protein 4 ( Samd4 ), a gene encoding an RNA-binding protein with no previously known function in mammals. This study provides evidence that Samd4 modulates the activities of the mechanistic target of rapamycin complex 1, a master regulator of metabolism.

Pharmacokinetic (PK) and pharmacodynamic (PD) modeling was conducted for the reduction of peripheral lymphocytes after oral administration of CS-0777 to healthy rats, monkeys and experimental autoimmune encephalomyelitis (EAE) induced rats. The phosphorylated active metabolite of CS-0777, M1, is a selective sphingosine 1-phosphate receptor-1 modulator. A linear one- and two-compartment model with a reversible metabolism process characterized the time courses of CS-0777 and M1 concentrations in rats and monkeys, respectively. The relationship between lymphocyte counts and M1 concentrations in blood was well described by an indirect response model in all animals examined. An I

Using an environmentally sensitized genetic screen we identified mutations that cause inflammatory colitis in mice. The X-linked Klein-Zschocher ( KLZ ) mutation created a null allele of Yipf6 , a member of a gene family believed to regulate vesicular transport in yeast, but without known functions in mammals. Yipf6 is a five transmembrane-spanning protein associated with Golgi compartments. Klein-Zschocher mutants were extremely sensitive to colitis induced by dextran sodium sulfate (DSS) and developed spontaneous ileitis and colitis after 16 mo of age in specific pathogen-free housing conditions. Electron microscopy, gene expression, and immunocytochemistry analyses provided evidence that impaired intestinal homeostasis stemmed from defective formation and secretion of large secretory granules from Paneth and goblet cells. These studies support a tissue- and organ-specific function for Yipf6 in the maintenance of intestinal homeostasis and implicate the orthologous human gene as a disease susceptibility locus.

We have previously disclosed 1,2,4-oxadiazole derivative 3 as a potent S1P3-sparing S1P1 agonist. Although compound 3 exhibits potent and manageable immunosuppressive efficacy in various in vivo models, recent studies have revealed that its 1,2,4-oxadiazole ring is subjected to enterobacterial decomposition. As provisions for unpredictable issues, a series of alternative compounds were synthesized on the basis of compound 3. Extensive SAR studies led to the finding of 1,3-thiazole 24c with the EC50 value of 3.4 nM for human S1P1, and over 5800-fold selectivity against S1P3. In rat on host versus graft reaction (HvGR), the ID50 value of 24c was determined at 0.07 mg/kg. The pharmacokinetics in rat and monkey is also reported. Compared to compound 3, 24c showed excellent stability against enterobacteria.

S1P(3)-sparing S1P(1) agonists have attracted attention as a suppressant of autoimmunity with reduced side effects. Our synthetic efforts and extensive SAR studies led to the discovery of 10b named CS-2100 with the EC(50) value of 4.0 nM for human S1P(1) and over 5000-fold selectivity against S1P(3). The in vivo immunosuppressive efficacy was evaluated in rats on host versus graft reaction and the ID(50) value was determined at 0.407mg/kg. The docking studies of CS-2100 with the homology model of S1P(1) and S1P(3) showed that the ethyl group on the thiophene ring of CS-2100 was sterically hindered by Phe263 in S1P(3), not in the case of Leu276 in S1P(1). This observation gives an explanation for the excellent S1P(3)-sparing characteristic of CS-2100.

CS-0777 (3) is phosphorylated in vivo, and the phosphate of CS-0777 (CS-0777-P) (4) acts as a selective S1P receptor-1 (S1P1) modulator. We report herein the synthesis of CS-0777 and CS-0777-P, pharmacological effects such as S1P1 and S1P3 agonist activity in vitro, peripheral blood lymphocyte lowering effects and the suppressive effect on experimental autoimmune encephalomyelitis (EAE), and also the pharmacokinetics in rats. CS-0777-P had ∼320-fold greater agonist activity for human S1P1 (EC50; 1.1 nM) relative to S1P3 (EC50; 350 nM). Following administration of single oral doses of 0.1 and 1 mg/kg of CS-0777 in rats, lymphocyte counts decreased significantly, with a nadir at 12 h postdose and recovery to vehicle control levels by 5 days postdose. In the EAE model compared to the vehicle-treated group, significant decreases in the cumulative EAE scores were observed for the 0.1 and 1 mg/kg CS-0777 groups in rats. CS-0777 is currently in clinical trials for the treatment of multiple sclerosis (MS).

We recently reported that nonsteroidal anti-inflammatory drug (NSAID)-induced gastric lesions involve NSAID-induced apoptosis of gastric mucosal cells, which in turn involves the endoplasmic reticulum stress response, in particular the up-regulation of CCAAT/enhancer-binding protein homologous transcription factor (CHOP). In this study, we have examined the molecular mechanism governing this NSAID-induced apoptosis in primary cultures of gastric mucosal cells. Various NSAIDs showed membrane permeabilization activity that correlated with their apoptosis-inducing activity. Various NSAIDs, particularly celecoxib, also increased intracellular Ca2+ levels. This increase was accompanied by K+ efflux from cells and was virtually absent when extracellular Ca2+ had been depleted. These data indicate that the increase in intracellular Ca2+ levels that is observed in the presence of NSAIDs is due to the stimulation of Ca2+ influx across the cytoplasmic membrane, which results from their membrane permeabilization activity. An intracellular Ca2+ chelator partially inhibited celecoxib-induced release of cytochrome c from mitochondria, reduced the magnitude of the celecoxib-induced decrease in mitochondrial membrane potential and inhibited celecoxib-induced apoptotic cell death. It is therefore likely that an increase in intracellular Ca2+ levels is involved in celecoxib-induced mitochondrial dysfunction and the resulting apoptosis. An inhibitor of calpain, a Ca2+-dependent cysteine protease, partially suppressed mitochondrial dysfunction and apoptosis in the presence of celecoxib. Celecoxib-dependent CHOP-induction was partially inhibited by the intracellular Ca2+ chelator but not by the calpain inhibitor. These results suggest that Ca2+-stimulated calpain activity and CHOP expression play important roles in celecoxib-induced apoptosis in gastric mucosal cells.

Prostaglandin E(2) (PGE(2)) has a strong protective effect on the gastric mucosa in vivo; however, the molecular mechanism of a direct cytoprotective effect of PGE(2) on gastric mucosal cells has yet to be elucidated. Although we reported previously that PGE(2) inhibited gastric irritant-induced apoptotic DNA fragmentation in primary cultures of guinea pig gastric mucosal cells, we show here that PGE(2) inhibits the ethanol-dependent release of cytochrome c from mitochondria. Of the four main subtypes of PGE(2) receptors, we also demonstrated, using subtype-specific agonists, that EP(2) and EP(4) receptors are involved in the PGE(2)-mediated protection of gastric mucosal cells from ethanol-induced apoptosis. Activation of EP(2) and EP(4) receptors is coupled with an increase in cAMP, for which a cAMP analogue was found here to inhibit the ethanol-induced apoptosis. The increase in cAMP is known to activate both protein kinase A (PKA) and phosphatidylinositol 3-kinase pathways. An inhibitor of PKA but not of phosphatidylinositol 3-kinase blocked the PGE(2)-mediated protection of cells from ethanol-induced apoptosis, suggesting that a PKA pathway is mainly responsible for the PGE(2)-mediated inhibition of apoptosis. Based on these results, we considered that PGE(2) inhibited gastric irritant-induced apoptosis in gastric mucosal cells via induction of an increase in cAMP and activation of PKA, and that this effect was involved in the PGE(2)-mediated protection of the gastric mucosa from gastric irritants in vivo.

In this study, we established a system of high concentration serum-dependent spontaneous apoptosis of guinea pig gastric pit cells in primary culture, which seems to mimic the spontaneous apoptosis of matured gastric pit cells at gastric surface in vivo. In addition to induction of the spontaneous apoptosis, cell growth was inhibited in the presence of 10% serum compared with 0.5% serum. Transforming growth factor-β1 (TGF-β1), which is known to cause both apoptosis and growth inhibition in mammalian cells, was present in serum of both fetal calf and guinea pig. The addition of recombinant TGF-β1 to the culture medium containing 0.5% fetal calf serum caused both induction of apoptosis and inhibition of cell growth. On the other hand, immunodepletion of TGF-β1 from fetal calf serum caused inability to induce both the spontaneous apoptosis and inhibition of cell growth. These data suggest that TGF-β1 is involved in the spontaneous apoptosis of guinea pig gastric pit cells in primary culture.

When the gastric mucosa is exposed to various irritants, apoptosis and subsequent gastric mucosal lesion can result in vivo. We here show that gastric irritants induced apoptosis in gastric mucosal cells in primary culture and examined its molecular mechanism. Ethanol, hydrogen peroxide, and hydrochloric acid all induced, in a dose-dependent manner, cell death, apoptotic DNA fragmentation, and chromatin condensation, suggesting that each of these gastric irritants induced apoptosis in vitro. Since each of these irritants decreased the mitochondrial membrane potential and stimulated the release of cytochrome c from mitochondria, gastric irritant-induced apoptosis seems to be mediated by mitochondrial dysfunction. Caspase-3, caspase-8, and caspase-9-like activities were all activated simultaneously by each of these irritants and the activation was concomitantly with cell death and apoptotic DNA fragmentation. Furthermore, pre-treatment of gastric mucosal cells with an inhibitor of caspase-8 suppressed the onset of cell death as well as the stimulation of caspase-3- and caspase-9-like activities caused by each of these gastric irritants. Based on these results, we consider that caspase-8, an initiator caspase, plays an important role in gastric irritant-induced apoptosis.

Various stressors induce apoptosis in gastric mucosal cells, which may cause gastric mucosal lesions in vivo. We recently reproduced gastric stressor-induced apoptosis in vitro, using primary cultures of guinea pig gastric mucosal cells. Geranylgeranylacetone is an antiulcer drug with heat-shock protein-inducing properties. The purpose of this study is to examine the effect of geranylgeranylacetone on gastric stressor-induced apoptosis in vitro. Ethanol, hydrogen peroxide, and hydrochloric acid all induced, in a dose-dependent manner, apoptotic DNA fragmentation. Pretreatment of cells with geranylgeranylacetone inhibited the apoptotic DNA fragmentation caused by each of these gastric stressors. Pretreatment of cells with a low concentration of ethanol, a procedure that is also known to induce heat-shock proteins, made cells resistant to the apoptotic DNA fragmentation. These results suggest that heat-shock proteins could be at least partly involved in the inhibitory effect of geranylgeranylacetone against apoptosis of gastric mucosal cells caused by these gastric stressors.

Guinea pig gastric pit cells in primary culture undergo serum-dependent cell maturation, which mimics their maturation in vivo. In this study we compared the sensitivities of these cells, as a function of early- and late-stage cell maturation, to various gastric stressors. Gastric pit cells in the early stage of maturation (two days of culture) were more resistant to apoptotic cell death induced by exposure to hydrogen peroxide than those cells in late-stage maturation (three days of culture). This maturation-associated difference was not observed for the sensitivities of cells to necrotic cell death induced by hydrogen peroxide, nor for necrotic and apoptotic cell death induced by other gastric stressors (ethanol and hydrochloric acid). The activities of catalase and glutathione peroxidase were specifically decreased in cells at the late stage of maturation compared to those at the early stage. The relative gastric pit cell phenotype sensitivity to hydrogen peroxide at the late stage of maturation may be due to a decrease in the activities of catalase and glutathione peroxidase, which are antioxidants for hydrogen peroxide.

Prostaglandins have cytoprotective effects on gastric mucosa via the influence of various mechanisms. The purpose of this study is to examine the effects of prostaglandins on maturation-dependent spontaneous apoptosis in gastric mucosal cells in vitro, which mimics the apoptosis of gastric mucosal cells related with a rapid cell turnover rate in vivo. Both prostaglandin E1 and E2 inhibited spontaneous apoptosis in a dose-dependent manner and increased the viability of gastric mucosal cells in culture. A number of antiulcer drugs presently in clinical use were shown to increase the concentrations of prostaglandins in cells. All of the drugs tested clearly inhibited the spontaneous apoptosis in a dose-dependent manner. Based on these results, we propose that the cytoprotective effects of prostaglandins on gastric mucosa in vivo can be partially explained by an increase in the number of gastric mucosal cells present as a result of the inhibition of maturation-dependent spontaneous apoptosis.

We previously reported that various gastric irritants induced both apoptosis and necrosis in cultured gastric mucosal cells. In a continuation of this work, the present study has examined the effects of prostaglandin E2 (PGE2), a cytoprotective factor for gastric mucosa in vivo, on gastric irritant-induced apoptosis and necrosis in vitro. PGE2 inhibited ethanol-induced apoptosis and increased cell viability in a dose-dependent manner in primary cultures of guinea pig gastric mucosal cells. PGE2 also inhibited hydrogen peroxide-induced apoptosis. In contrast, PGE2 showed no cytoprotective effects against ethanol-induced necrosis. Based on these results, we consider that the cytoprotective effects of PGE2 on gastric mucosa in vivo can be partially explained by its inhibitory effect on gastric irritant-induced apoptosis.

A major clinical problem encountered with the use of nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin is gastropathy. In this study, we examined, using guinea pig gastric mucosal cells in primary culture, how NSAIDs damage gastric mucosal cells. The short-term treatment of cells with high concentrations of indomethacin decreased cell viability in the absence of apoptotic DNA fragmentation, chromatin condensation, or caspase activation. Cells lost membrane integrity with this short-term indomethacin treatment, suggesting that indomethacin induced necrosis under these conditions. In contrast, the long-term treatment of cells with low concentrations of indomethacin decreased cell viability and was accompanied by apoptotic DNA fragmentation, chromatin condensation, and caspase activation. Pretreatment of cells with inhibitors of caspases or protein synthesis suppressed cell death caused by long-term indomethacin treatment, suggesting that apoptosis was induced when the inhibitors were not present. These results imply that NSAIDs cause gastric mucosal damage through both necrosis and apoptosis of gastric mucosal cells.

Gastric mucosal cell death due to various gastric stressors can cause several types of gastric diseases, such as gastric ulcers. In this study, we examined cell death following the short-term treatment of guinea pig gastric mucosal cells in primary culture with various gastric stressors. The short-term treatment of cells with ethanol, hydrogen peroxide or hydrochloric acid caused, in a dose-dependent manner, cell death in the absence of apoptotic DNA fragmentation and chromatin condensation. Cells lost membrane integrity following the treatment with each of these gastric stressors, suggesting that necrosis was induced in gastric mucosal cells by short-term treatment of the cells with gastric stressors. Geranylgeranylacetone, an anti-ulcer drug with heat-shock protein inducing properties, protected gastric mucosal cells from the necrotic cell death caused by each of these gastric stressors. Pretreatment of cells with low concentrations of ethanol (3%), which also induced heat-shock protein, made cells resistant to the necrotic cell death caused by the gastric stressors. These results suggest that heat-shock proteins is involved in the cytoprotective effect of geranylgeranylacetone against necrotic cell death.

One of the major side effects of nonsteroidal antiinflammatory drugs, such as indomethacin, is gastropathy. The purpose of this study was to search for a therapeutic drug to prevent this side effect in vitro. We found that geranylgeranylacetone, a unique antiulcer drug with a heat-shock protein-inducing ability, protected cultured guinea pig gastric mucosal cells from cell damage caused by indomethacin. This cytoprotective effect of geranylgeranylacetone required concentrations of more than 10−6 M and incubation periods of longer than 2 hr. Pretreatment of cells with an inhibitor of protein synthesis completely abolished the cytoprotective effect of geranylgeranylacetone, suggesting that some proteins induced by the drug are responsible for the cytoprotection. Since pretreatment of cells with low concentrations of ethanol, which also induced the heat-shock proteins, made cells resistant to indomethacin, heat-shock proteins are candidates for the proteins that are involved in the cytoprotective effect of geranylgeranylacetone against indomethacin.

TNFα is a powerful inflammatory stimulus, central both to the control of infection, and as an agent of inflammatory disease. The most potent inducers of TNFα secretion signal through the Toll-like receptors, and we describe here a chemically-induced mutation that impairs this response in macrophages. A missense mutation was revealed in the gene encoding the inactive rhomboid protease iRhom2, which was not complemented by a null allele of the same gene. Neither the missense nor the null allele affected TLR-induced secretion of IL-6. Moreover, unlike a mutation in TNFα, the iRhom2 missense mutation did not cause enhanced susceptibility to colitis induced by dextran sodium sulfate. These results establish a specific role for iRhom2 in the secretion of TNFα, and present a new target for the modulation of inflammation.

We have previously disclosed 1,2,4-oxadiazole derivative 3 as a potent S1P(3)-sparing S1P(1) agonist. Although compound 3 exhibits potent and manageable immunosuppressive efficacy in various in vivo models, recent studies have revealed that its 1,2,4-oxadiazole ring is subjected to enterobacterial decomposition. As provisions for unpredictable issues, a series of alternative compounds were synthesized on the basis of compound 3. Extensive SAR studies led to the finding of 1,3-thiazole 24c with the EC(50) value of 3.4 nM for human S1P(1), and over 5800-fold selectivity against S1P(3). In rat on host versus graft reaction (HvGR), the ID(50) value of 24c was determined at 0.07 mg/kg. The pharmacokinetics in rat and monkey is also reported. Compared to compound 3, 24c showed excellent stability against enterobacteria.

Background We present a compendium of N-ethyl-N-nitrosourea (ENU)-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1) to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2) to assess the characteristics of these mutations; and 3) to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype. Findings In the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied. Conclusions Collectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by amino acid substitutions, and may lead to refined predictions as to whether specific amino acid changes are responsible for observed phenotypes. These data form the basis for closer in silico estimations of the number of genes mutated to a state of phenovariance by ENU within a population of G3 mice.

Modulators of sphingosine phosphate receptor-1 (S1P(1)) have recently been focused as a suppressant of autoimmunity. We have discovered a 4-ethylthiophene-based S1P(1) agonist 1-({4-Ethyl-5-[5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl]-2-thienyl}methyl)azetidine-3-carboxylic acid (CS-2100, 8) showing potent S1P(1) agonist activity against S1P(3) and an excellent in vivo potency. We report herein the synthesis of CS-2100 (8) and pharmacological effects such as S1P(1) and S1P(3) agonist activity in vitro, peripheral blood lymphocyte lowering effects and the suppressive effects on adjuvant-induced arthritis and experimental autoimmune encephalomyelitis (EAE) in animal models. The pharmacokinetic data were also reported. CS-2100 (8) had >5000-fold greater agonist activity for human S1P(1) (EC(50); 4.0 nM) relative to S1P(3) (EC(50); >20,000 nM). Following administration of single oral doses of 0.1 and 1 mg/kg of CS-2100 (8) in rats, lymphocyte counts decreased significantly, with a nadir at 8 and/or 12 h post-dose and recovery to vehicle control levels by 24-48 h post-dose. CS-2100 (8) is efficacious in the adjuvant-induced arthritis model in rats (ID(50); 0.44 mg/kg). In the EAE model compared to the vehicle-treated group, significant decreases in the cumulative EAE scores were observed for 0.3 and 1 mg/kg CS-2100 (8) groups in mice. While CS-2100 (8) showed potent efficacy in various animal disease models, it was also revealed that the central 1,2,4-oxadiazole ring of CS-2100 (8) was decomposed by enterobacteria in intestine of rats and monkeys, implicating the latent concern about an external susceptibility in its metabolic process in the upcoming clinical studies.

Toll-like receptors (TLRs) trigger intestinal inflammation when the epithelial barrier is breached by physical trauma or pathogenic microbes. Although it has been shown that TLR-mediated signals are ultimately protective in models of acute intestinal inflammation [such as dextran sulfate sodium (DSS)-induced colitis], it is less clear which cells mediate protection. Here we demonstrate that TLR signaling in the nonhematopoietic compartment confers protection in acute DSS-induced colitis. Epithelial cells of MyD88/Trif-deficient mice express diminished levels of the epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG), and systemic lipopolysaccharide administration induces their expression in the colon. N -ethyl- N -nitrosourea (ENU)-induced mutations in Adam17 (which is required for AREG and EREG processing) and in Egfr both produce a strong DSS colitis phenotype, and the Adam17 mutation exerts its deleterious effect in the nonhematopoietic compartment. The effect of abrogation of TLR signaling is mitigated by systemic administration of AREG. A TLR→MyD88→AREG/EREG→EGFR signaling pathway is represented in nonhematopoietic cells of the intestinal tract, responds to microbial stimuli once barriers are breached, and mediates protection against DSS-induced colitis.

Nonsteroidal anti-inflammatory drugs (NSAID) have shown chemopreventive effects in both preclinical and clinical studies; however, the precise molecular mechanism governing this response remains unclear. We used DNA microarray techniques to search for genes whose expression is induced by the NSAID indomethacin in human gastric carcinoma (AGS) cells. Among identified genes, we focused on those related to tight junction function (claudin-4, claudin-1, and occludin), particularly claudin-4. Induction of claudin-4 by indomethacin was confirmed at both mRNA and protein levels. NSAIDs, other than indomethacin (diclofenac and celecoxib), also induced claudin-4. All of the tested NSAIDs increased the intracellular Ca2+ concentration. Other drugs that increased the intracellular Ca2+ concentration (thapsigargin and ionomycin) also induced claudin-4. Furthermore, an intracellular Ca2+ chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid] inhibited the indomethacin-dependent induction of claudin-4. These results strongly suggest that induction of claudin-4 by indomethacin is mediated through an increase in the intracellular Ca2+ concentration. Overexpression of claudin-4 in AGS cells did not affect cell growth or the induction of apoptosis by indomethacin. On the other hand, addition of indomethacin or overexpression of claudin-4 inhibited cell migration. Colony formation in soft agar was also inhibited. Suppression of claudin-4 expression by small interfering RNA restored the migration activity of AGS cells in the presence of indomethacin. Based on these results, we consider that the induction of claudin-4 and other tight junction–related genes by NSAIDs may be involved in the chemopreventive effect of NSAIDs through the suppression of anchorage-independent growth and cell migration.

Nitric oxide (NO) releasing non-steroidal anti-inflammatory drugs (NSAIDs) have shown a marked reduction of gastrointestinal side effects and we here examined the cytotoxicity of NCX 530 (NO-indomethacin). Under conditions where indomethacin clearly induced both necrosis and apoptosis, NCX 530 induced neither. NCX 530 protected cells from celecoxib-induced necrosis and apoptosis. NCX 530 partially suppressed celecoxib-dependent membrane permeabilization and an inhibitor for guanylate cyclase suppressed the cytoprotective effect of NCX 530 against celecoxib. In vivo, NCX 530 alone produced fewer gastric lesions in rats than did indomethacin. A combination of the oral administration of celecoxib together with the intraperitoneal administration of indomethacin, but not of NCX 530, clearly resulted in the production of gastric lesions. The low direct cytotoxicity and the cytoprotective effect of NCX 530 observed in vitro may also act in vivo, thus ensuring that NCX 530 is safe for use on the gastric mucosa.

Prodrugs of non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for clinical purposes because they are not harmful to the gastrointestinal mucosa. We recently showed that NSAIDs have direct cytotoxicity in NSAID-induced gastric lesions. We show here that under conditions where the NSAIDs indomethacin and celecoxib clearly induce cell death, an NSAID prodrug, nabumetone, and its active metabolite 6-methoxy-2-naphthylacetic acid (6MNA), did not have such effects. Moreover, nabumetone and 6MNA exhibited much lower membrane permeabilizing activities than did indomethacin and celecoxib. We recently reported that when an orally administered NSAID was used in combination with a low dose of intravenously administered indomethacin, the severity of gastric lesions produced in rats depended on the cytotoxicity of the orally administered NSAID. Using a similar protocol, we show here that gastric lesions were produced when the orally administered NSAID was celecoxib, but not when nabumetone was used. We thus propose that the low direct cytotoxicity of nabumetone observed in vitro is maintained in vivo, and that the use of nabumetone does not harm the gastric mucosa.

The cytotoxicity of non-steroidal anti-inflammatory drugs (NSAIDs) is involved in the formation of NSAID-induced gastric lesions. The mechanism(s) behind these cytotoxic effects, however, is not well understood. We found here that several NSAIDs tested caused hemolysis when employed at concentrations similar to those that result in cytotoxicity. Moreover, these same NSAIDs were found to directly permeabilize the membranes of calcein-loaded liposomes. Given the similarity in NSAID concentrations for cytotoxic and membrane permeabilization effects, the cytotoxic action of these NSAIDs may be mediated through the permeabilization of biological membranes. Increase in the intracellular Ca(2+) level can lead to cell death. We here found that all of NSAIDs tested increased the intracellular Ca(2+) level at concentrations similar to those that result in cytotoxicity. Based on these results, we consider a possibility that membrane permeabilization by NSAIDs induces cell death through increase in the intracellular Ca(2+) level.

Orally ingested non-steroidal anti-inflammatory drugs (NSAIDs) and acid in gastric secretions are gastric irritants that co-exist at the surface of the gastric mucosa. Here, we examined the individual and combined effects of indomethacin, a typical NSAID, and hydrochloric acid on cell death in primary cultures of guinea pig gastric mucosal cells. Indomethacin alone (at concentrations less than 200 microM) did not induce apoptosis; however, hydrochloric acid-induced apoptosis was stimulated in the presence of indomethacin (50-200 microM). Isobologram analysis confirmed the presence of a cytotoxic synergy between indomethacin and hydrochloric acid. The synergistic response between the two gastric irritants was also observed for necrosis. Given that the IC50 value of indomethacin for inhibition of prostaglandin synthesis is about 5 nM, the synergistic response between indomethacin and hydrochloric acid appears to be independent of the inhibition of cyclooxygenase activity by indomethacin.

Apoptosis induced by nonsteroidal anti-inflammatory drugs (NSAIDs) is involved not only in the production of NSAID-induced gastric lesions but also in the antitumor activity of these drugs. The endoplasmic reticulum (ER) stress response is a cellular mechanism that aids in protecting the ER against ER stressors and is involved in ER stressor-induced apoptosis. Here, we examine the relationship between this response and NSAID-induced apoptosis in cultured guinea-pig gastric mucosal cells. Exposure of cells to indomethacin, a commonly used NSAID, induced GRP78 as well as CHOP, a transcription factor involved in apoptosis. Three factors that positively regulate CHOP expression (ATF6, ATF4 and XBP-1) were activated and/or induced by indomethacin. NSAIDs other than indomethacin (diclofenac, ibuprofen and celecoxib) also induced CHOP. Monitoring of the transcriptional activities of ATF6 and CHOP by luciferase assay revealed that both were stimulated in the presence of indomethacin. Furthermore, indomethacin-induced apoptosis was suppressed in cultured guinea-pig gastric mucosal cells by expression of the dominant-negative form of CHOP, or in peritoneal macrophages from CHOP-deficient mice. These results suggest that ER stress response-related proteins, particularly CHOP, are involved in NSAID-induced apoptosis.

In this study, we examined adaptive cytoprotection against NSAIDs in human gastric carcinoma cells in culture. Pretreatment of cells with low (nontoxic) concentrations of ethanol protected cells from cell death induced by subsequent exposure to NSAIDs. The adaptive cytoprotection against NSAIDs induced by ethanol was not attenuated by pretreatment of cells with inhibitors of protein synthesis or prostaglandin synthesis, thus inferring that neither newly synthesized proteins nor prostaglandins are involved in this process. Furthermore, treatment of cells with the low concentration of ethanol did not affect the synthesis and secretion of mucin. In in vivo experiments on rats, oral preadministration of a low dose of ethanol protected the gastric mucosa from gastric lesions induced by subsequent oral administration of NSAIDs. One possible explanation for this in vivo phenomenon is that the adaptive cytoprotection induced by ethanol protects the gastric mucosa from the direct cytotoxic effect of NSAIDs.

A major clinical problem encountered with the use of non-steroidal anti-inflammatory drugs (NSAIDs), is gastrointestinal complications. We previously reported that NSAIDs induce both necrosis and apoptosis in vitro. We here examined the cyclooxygenase (COX) dependency of this cytotoxic effect of NSAIDs and its involvement in NSAID-induced gastric lesions. Necrosis and apoptosis by NSAIDs was observed with all selective COX-2 inhibitors except rofecoxib and was not inhibited by exogenously added prostaglandin E2, suggesting that cytotoxicity of NSAIDs seems to be independent of the inhibition of COX. Intravenously administered indomethacin, which completely inhibited COX activity at gastric mucosa, did not produce gastric lesions. Orally administered selective COX-2 inhibitors, which did not inhibit COX at gastric mucosa, also did not produce gastric lesions. Interestingly, a combination of the oral administration of each of all selective COX-2 inhibitors except rofecoxib with the intravenous administration of indomethacin clearly produced gastric lesions. These results suggest that in addition to COX inhibition by NSAIDs, direct cytotoxicity of NSAIDs may be involved in NSAID-induced gastric lesions.

We previously reported that various gastric irritants induced both apoptosis and necrosis in cultured gastric mucosal cells. In a continuation of this work, the present study has examined the effects of prostaglandin E2 (PGE2), a cytoprotective factor for gastric mucosa in vivo, on gastric irritant-induced apoptosis and necrosis in vitro. PGE2 inhibited ethanol-induced apoptosis and increased cell viability in a dose-dependent manner in primary cultures of guinea pig gastric mucosal cells. PGE2 also inhibited hydrogen peroxide-induced apoptosis. In contrast, PGE2 showed no cytoprotective effects against ethanol-induced necrosis. Based on these results, we consider that the cytoprotective effects of PGE2 on gastric mucosa in vivo can be partially explained by its inhibitory effect on gastric irritant-induced apoptosis.

Guinea pig gastric pit cells in primary culture undergo serum-dependent cell maturation, which mimics their maturation in vivo. In this study we compared the sensitivities of these cells, as a function of early- and late-stage cell maturation, to various gastric stressors. Gastric pit cells in the early stage of maturation (two days of culture) were more resistant to apoptotic cell death induced by exposure to hydrogen peroxide than those cells in late-stage maturation (three days of culture). This maturation-associated difference was not observed for the sensitivities of cells to necrotic cell death induced by hydrogen peroxide, nor for necrotic and apoptotic cell death induced by other gastric stressors (ethanol and hydrochloric acid). The activities of catalase and glutathione peroxidase were specifically decreased in cells at the late stage of maturation compared to those at the early stage. The relative gastric pit cell phenotype sensitivity to hydrogen peroxide at the late stage of maturation may be due to a decrease in the activities of catalase and glutathione peroxidase, which are antioxidants for hydrogen peroxide.

Various stressors induce apoptosis in gastric mucosal cells, which may cause gastric mucosal lesions in vivo. We recently reproduced gastric stressor-induced apoptosis in vitro, using primary cultures of guinea pig gastric mucosal cells. Geranylgeranylacetone is an antiulcer drug with heat-shock protein-inducing properties. The purpose of this study is to examine the effect of geranylgeranylacetone on gastric stressor-induced apoptosis in vitro. Ethanol, hydrogen peroxide, and hydrochloric acid all induced, in a dose-dependent manner, apoptotic DNA fragmentation. Pretreatment of cells with geranylgeranylacetone inhibited the apoptotic DNA fragmentation caused by each of these gastric stressors. Pretreatment of cells with a low concentration of ethanol, a procedure that is also known tb induce heat-shock proteins, made cells resistant to the apoptotic DNA fragmentation. These results suggest that heat-shock proteins could be at least partly involved in the inhibitory effect of geranylgeranylacetone against apoptosis of gastric mucosal cells caused by these gastric stressors.

Prostaglandins have cytoprotective effects on gastric mucosa via the influence of various mechanisms. The purpose of this study is to examine the effects of prostaglandins on maturation-dependent spontaneous apoptosis in gastric mucosal cells in vitro, which mimics the apoptosis of gastric mucosal cells related with a rapid cell turnover rate in vivo. Both prostaglandin E1 and E2 inhibited spontaneous apoptosis in a dose-dependent manner and increased the viability of gastric mucosal cells in culture. A number of antiulcer drugs presently in clinical use were shown to increase the concentrations of prostaglandins in cells. All of the drugs tested clearly inhibited the spontaneous apoptosis in a dose-dependent manner. Based on these results, we propose that the cytoprotective effects of prostaglandins on gastric mucosa in vivo can be partially explained by an increase in the number of gastric mucosal cells present as a result of the inhibition of maturation-dependent spontaneous apoptosis.